The purpose of this project is to define hemostatic regulatory mechanisms of the brain. This will be investigated from two-perspectives: an in vitro analysis of the hemostatic effects of astrocyte-endothelial interactions, and an analysis of fibrinolytic proteins in post-mortem brain tissue. The in vitro study will utilize bovine brain capillary endothelial cells grown in monolayers and capillary-like structures, and will define the hemostatic effects of a) soluble factors elaborated by astrocytes and b) direct contact of astrocytes with capillary-like structures. Hemostatic factors to be studied in vitro include thrombomodulin, tissue factor, tissue plasminogen activator (tPA), and plasminogen activator inhibitor-1 (PAI-1). Analysis of hemostasis factors will use antigenic and functional assays, as well as polymerase chain reaction and in-situ hybridization techniques. Neutralizing antibodies will be used to define the roles of interleukin-l (IL-I) and tumor necrosis factor (TNF) as mediators of astrocyte effects on endothelial hemostasis. The relationship between fibrinolysis and the brain will be further studied using autopsy samples to analyze the regional distribution of endothelial fibrinolytic proteins tPA, urokinase-type plasminogen activator (uPA), and PAI-1. This will be studied in brain from subjects without stroke risk factors, subjects with hypertension, and subjects with diabetes. The relationship between brain fibrinolysis, stroke risk factors, and brain expression of IL-1 and TNF will be defined. Vascular expression of fibrinolytic factors will be quantified using immunocytochemistry and tissue content will be determined by enzyme immunoassay; in-situ hybridization will also be used for localization purposes. Completion of this project will substantially enhance our knowledge of processes relevant to ischemic stroke pathogenesis and lead to new strategies for stroke prevention.